Biosynthesis of phloroglucinol and preparation of 1,3-dihydroxybenzene therefrom

ABSTRACT

The present invention provides methods, enzymes, and cells for the biosynthetic production of phloroglucinol from malonyl-CoA, which is ultimately obtained from simple starting materials such as glucose; also provided are methods for preparing derivatives of biosynthetic phloroglucinol, including, e.g., resorcinol.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of International ApplicationNo. PCT/US2005/036291, filed Oct. 11, 2005, which claims the benefit ofU.S. Provisional Application Ser. No. 60/617,959, and of U.S.Provisional Application Ser. No. 60/618,024, both filed Oct. 12, 2004,the entire contents of which are hereby incorporated by reference intothe present application.

STATEMENT OF GOVERNMENT INTEREST

This invention was made in part with Government support under Grant No.N00014-02-1-0725, awarded by the Office of Naval Research. Thegovernment may have certain rights in this invention.

BACKGROUND

Phloroglucinol (1,3,5-trihydroxybenzene) and its derivatives are widelyused in commerce. Phloroglucinol and its derivatives, e.g.,trimethylphloroglucinol, are used as pharmaceutical agents, e.g., asantispasmodics. Phloroglucinol is used as a starting material orintermediate in pharmaceutical, microbicide, and other organicsyntheses. Phloroglucinol is used as a stain for microscopy samples thatcontain lignin (e.g., wood samples), and it is used in the manufactureof dyes, including leather, textile, and hair dyes. It is used in themanufacture of adhesives and as an epoxy resin curing agent, and in thepreparation of explosives, e.g., the thermally- and shock-stable highexplosive, 1,3,4-triamino-2,4,6-trinitrobenzene (TATB). Phloroglucinolalso functions as an antioxidant, stabilizer, and corrosion resistanceagent, and is utilized as a coupling agent for photosensitiveduplicating paper, as a substitute for silver iodide in rain-making, asa bone sample decalcifying agent, and as a floral preservative.Phloroglucinol can also be converted to resorcinol by catalytichydrogenation.

Resorcinol (1,3-dihydroxybenzene) is a particularly useful derivative ofphloroglucinol, although resorcinol is not currently produced by thatroute. As is phloroglucinol, resorcinol is used in the manufacture ofdyes and adhesives, and as an epoxy resin curing agent; and it is usedas a starting material or intermediate in pharmaceutical and otherorganic syntheses. Resorcinol and its derivatives are further commonlyused, either alone or with other active ingredients such as sulfur, incosmetics and in topical skin medicaments for treatment of conditionsincluding acne, dandruff, eczema, and psoriasis, functioning, in part,as an antiseptic and antipruritic. Resorcinol is also used as across-linking agent for neoprene, as a tack-enhancing agent in rubbercompositions, in bonding agents for organic polymers (e.g., melamine andrubber), and in the fabrication of fibrous and other compositematerials. Resorcinol is used: in the manufacture of resins and resinadhesives, e.g., both as a monomer and as a UV absorbing agent; in themanufacture of explosives, e.g., energetic compounds such as styphnicacid (2,4,6-trinitrobenzene-1,3-diol) and heavy metal styphnates; and inthe synthesis of diazo dyes, plasticizers, hexyl resorcinol, andp-aminosalicylic acid.

The most common of the resorcinol-based resins are resorcinol-aldehydeand resorcinol-phenol-aldehyde resins. These types of resorcinol-basedresins are used, for example, as resin adhesives, composite materialmatrices, and as starting materials for rayon and nylon production.Examples of composite materials include resorcinol-formaldehyde carbon(or other organic) particle hydrogels, aerogels, and xerogels, which areuseful, e.g., as matrix materials for metallic and organometalliccatalysts. Resorcinol-formaldehyde resins and particulate compositestherewith are also used in dentistry as a root canal filling material.

Resorcinol-aldehyde resin adhesives are especially useful inapplications requiring high bond strength, including, e.g.: woodentrusses, joists, barrels, and boats; and aircraft. Modifiedresorcinol-aldehyde resin adhesives are also used as biological woundsealant compositions both on topical wounds and on internal wounds orsurgical cuts, e.g., vascular incisions. This is often done in militaryfield medicine, e.g., to minimize environmental exposure, reducebleeding and fluid loss, and speed the healing process. Such modifiedresin adhesives include, e.g., gelatin-resorcinol-formaldehyde andgelatin-resorcinol-glutaraldehyde compositions, wherein the aldehyde maybe maintained separately from, and later mixed with, theresorcinol-gelatin composition to form the sealant when needed.

Currently, both phloroglucinol and resorcinol are commercially producedby chemical organic synthesis using caustics and high temperatures,beginning with petroleum-derived starting materials and creating muchenvironmentally problematic waste.

As a result, it would be an improvement in the art to provide moreefficient and cleaner processes for the production of these valuablecompounds. One possible solution might be to provide a biosyntheticroute for production of phloroglucinol, with an optional hydrogenationof the biosynthetic phloroglucinol to resorcinol. Biosyntheticproduction of compounds related to phloroglucinol has been reported inplants, algae, and microbes, e.g.: acetyl phloroglucinols fromPseudomonas spp.; hyperforins, hyperfoliatins, hyperjovinols, andhyperatomarins from Hypericum spp.; pallidusol, dehydropallidusol,pallidol, mallopallidol, and homomallopallidol from Mallotus spp.;garcinielliptones from Garcinia spp.; flavaspidic acids from Dryopterisspp.; macrocarpals and sideroxylonals from Eucalyptus spp.;1,3,5-trimethoxybenzene from Rosa spp.; as well asphloroglucinol-containing glycosides and phlorotannins.

However, production of phloroglucinol is reported in such plants andmicrobes as merely a degradation product of more complex, and thus lessabundant and/or more costly, starting materials. See, e.g.: L. Schoeferet al., Appl. Environ. Microbiol. 70(10):6131-37 (2004); D. Baas & J.Rétey, Eur. J. Biochem. 265:896-901 (1999). In addition, microbialbiosynthetic production of di-acetyl phloroglucinols has been proposedas a means for improving the anti-fungal activity of recombinantbacteria to be released into the agricultural environment as biocontrolagents against phytopathogens. See U.S. Pat. No. 6,051,383, Thomashow etal., issued Apr. 18, 2000; and M. G. Bangera & L. S. Thomashow, J Bact.181(10):3155-63 (1999). Yet, a route of anabolic biosynthetic productionof phloroglucinol, e.g., from inexpensive starting materials such asglucose, is not shown.

Recently, an alternate route (see FIG. 2) to phloroglucinol (1a) hasbeen elaborated, which involves microbe-catalyzed synthesis of triaceticacid lactone (3a) from glucose; however, it has been found that multiplechemical steps are needed to convert triacetic acid lactone (3a) intophloroglucinol (1a). See W. Zha et al., J. Am. Chem. Soc.126(14):4534-35 (2004); and C. A. Hansen & J. W. Frost, J. Am. Chem.Soc. 124(21):5926-27 (2002). Thus, this route is at best a partlybiosynthetic, partly chemosynthetic pathway.

Thus, to date, no fully biosynthetic route useful for commercialproduction of phloroglucinol per se (1,3,5-trihydroxybenzene) has beenreported. No enzymes or encoding genes that catalyze the formation ofphloroglucinol per se have been identified.

SUMMARY

The present invention provides methods, enzymes, and cells for thebiosynthetic production of phloroglucinol from malonyl-CoA, andultimately from simple starting materials such as glucose. Specifically,the present invention provides the first entirely biosynthetic, anabolicroute for phloroglucinol synthesis that does not require all four of thephlABCD operon enzymes, but is capable of commercial phloroglucinolproduction using only a phlD enzyme or other phloroglucinol synthase.Also provided are methods for preparing derivatives of biosyntheticphloroglucinol, including, e.g., resorcinol. Uses of the enzyme systems,recombinant cells, and methods, for production of phloroglucinol;phloroglucinol produced thereby. Uses of the enzyme systems, recombinantcells, and methods, for production of phloroglucinol derivative(s),e.g., resorcinol; phloroglucinol and derivatives, e.g., resorcinol,produced thereby. Uses of the enzyme systems, recombinant cells,methods, phloroglucinol, or derivative(s) for production of compounds orcompositions, e.g., explosive or propellant compounds and compositions;uses of the enzyme systems, recombinant cells, methods, phloroglucinol,or derivative(s) for production of non-explosive, non-propellantcompounds and compositions, such as medicament, cosmetic, dye, polymerresin, rubber, adhesive, sealant, coating, composite material, orlaminated or bonded materials. Explosive or propellant compounds andcompositions produced thereby; non-explosive, non-propellant compoundsand compositions produced thereby. The present invention furtherprovides:

Isolated or recombinant PhlD⁺ enzyme systems that are at least one ofPhlA⁻, PhlB⁻, or PhlC⁻; PhlD⁺ recombinant cells that are at least one ofPhlA⁻, PhlB⁻, or PhlC⁻; and PhlD⁺ recombinant cells that have beengenetically engineered to increase the expression of PhlD therein; whichenzyme systems and recombinant cells are capable of convertingmalonyl-CoA to phloroglucinol; such enzyme systems and cells that arePhlA⁻, PhlB⁻, and PhlC⁻; such enzyme systems and cells that furthercomprise at least one malonyl-CoA synthesis enzyme;

Processes for production of anabolic phloroglucinol involving providingsuch an enzyme system or recombinant cell and malonyl-CoA or anothercarbon source that the enzyme system or recombinant cell is capable ofconverting to malonyl-CoA, contacting either the malonyl-CoA with theenzyme system or recombinant cell under conditions in which it cansynthesize phloroglucinol therefrom, or the other carbon source with theenzyme system or recombinant cell under conditions in which it canconvert the carbon source to malonyl-CoA and can synthesizephloroglucinol therefrom; such processes in which the carbon source is asimple carbon source, such as saccharide, an aliphatic polyol, or acombination thereof; such processes in which the cell is cultured in amedium containing the carbon source, or where the culturing is performedas an extractive fermentation, and/or where the culturing utilizes amulti-temperature profile, such as a dual-temperature profile;phloroglucinol prepared by such processes;

Isolated or recombinant phloroglucinol synthase enzymes capable ofconverting malonyl-CoA to phloroglucinol; such enzymes comprising anamino acid sequence of SEQ ID NO:2, or an amino acid sequence that is atleast 70% homologous to SEQ ID NO:2, such as a conservativelysubstituted variant of the amino acid sequence of SEQ ID NO:2;

Isolated or recombinant nucleic acids comprising at least one openreading frame encoding a PhlD enzyme, and that is at least one of phlA⁻,phlB⁻, or phlC⁻; such nucleic acids further comprises at least one openreading frame encoding a malonyl-CoA synthesis enzyme; such nucleicacids in which the PhlD-encoding ORF comprises a base sequence of, or atleast 80% homologous to, SEQ ID NO:1, an RNA base sequence correspondingthereto, or a codon sequence redundant therewith;

phlD⁺ recombinant cells that have been transformed with such a nucleicacid, from which the cell can express phloroglucinol synthase; suchcells that are at least one of phlA⁻, phlB⁻, or phlC⁻; such cells thatare phlA⁻, phlB⁻, and phlC⁻; such cells that are further at least one ofphlE⁻ or phlF;

Processes for the preparation of resorcinol involving providing suchanabolic phloroglucinol biosynthesized by such an isolated orrecombinant enzyme system or recombinant cells, along with hydrogen anda rhodium catalyst, and contacting the phloroglucinol with the hydrogenand the rhodium catalyst under conditions in which the phloroglucinol ishydrogenated to form resorcinol; resorcinol prepared from such anabolicphloroglucinol;

Uses of such anabolic phloroglucinol, or of resorcinol preparedtherefrom, in the manufacture of a medicament, cosmetic, dye, polymerresin, rubber, adhesive, sealant, coating, composite material, orlaminated or bonded material; medicament, cosmetic, dye, polymer resin,rubber, adhesive, sealant, coating, composite material, or laminated orbonded material composition containing, or resulting from a chemicalmodification of, such anabolic phloroglucinol, or of resorcinol preparedtherefrom;

Processes for the preparation of recombinant cells that are capable ofbiosynthesizing phloroglucinol from malonyl-CoA, involving transforminga cell with such a PhlD-encoding nucleic acid that is capable ofexpression by the cell, or involving inactivating genes in a phlD⁺ cellby providing a phlD⁺ cell that is at least one of phlA⁺, phlB⁺, orphlC⁺, and inactivating at least one of the phlA, phlB, or phlC genestherein; such recombinant cells that are microbes, such as bacteria,examples of which include Escherichia coli and Pseudomonas fluorescens;such processes in which the enzyme system or recombinant cell comprisesa malonyl-CoA synthesis enzyme; such processes involving providing aphlABCD⁺ cell, and either or both of inactivating at least one phlA,phlB, or phlC gene thereof or inserting at least one phlD⁺ nucleic acidtherein that is at least one of phlA⁻, phlB⁻, or phlC⁻; such processesin which the phlD⁺ recombinant cell is phlA⁻, phlB⁻, and phlC⁻; suchprocesses involving providing a phlABCD⁺ cell, and inactivating all ofthe phlA, phlB, and phlC genes thereof; such processes involvingproviding a phlABCD⁻ cell and inserting a phlD gene therein; suchprocesses in which the phlD⁺ nucleic acid is located in genomic DNA ofthe cell; such processes in which it is located in extra-genomic DNA ofthe cell.

Methods for producing propellant or explosive compounds involvingproviding anabolic phloroglucinol biosynthesized by such an isolated orrecombinant enzyme system or recombinant cells and chemically modifyingthe anabolic phloroglucinol, or chemically modifying resorcinol preparedtherefrom;

Uses of such anabolic phloroglucinol, or of resorcinol preparedtherefrom, in the manufacture of an explosive or propellant; andexplosive or propellant compositions containing, or resulting from achemical modification of, such anabolic phloroglucinol, or of resorcinolprepared therefrom.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 presents Scheme 1, which illustrates literature-reported routes:(a) for acetylphloroglucinol biosynthesis without phloroglucinol as anintermediate, see M. G. Bangera & L. S. Thomashow, J Bact.181(10):3155-63 (1999); and (c) for triacetic acid lactone biosynthesis,see S. Eckermann et al., Nature 396:387 (1998), J. M. Jez et al., Chem.Bio. 7:919 (2000); W. Zha et al., J. Am. Chem. Soc. 126:4534 (2004).Also shown are the routes (b) and (b′) postulated and identified in thepresent work for acetylphloroglucinol biosynthesis with phloroglucinolas an intermediate.

FIG. 2 presents Scheme 2, which illustrates: the common commercialchemical synthetic route (a, b, c) for phloroglucinol synthesis; amulti-step route (d, e, f, g) previously proposed for synthesis ofphloroglucinol from glucose; a first, common commercial chemicalsynthetic route (i, j) for resorcinol synthesis; and a second, commoncommercial chemical synthetic route (k, l) for resorcinol synthesis.Also illustrated with circled arrows are: (1) the fully biosyntheticroute reported in the present work (indicated by a circled asterisk) forproduction of phloroglucinol; and (2) the chemical hydrogenation (h) ofphloroglucinol to resorcinol. Specific reactions or reaction steps shownare: (a) Na₂Cr₂O₇, H₂SO₄; (b) Fe, HCl; (c) H₂SO₄, 108° C.; (d) see W.Zha et al., J. Am. Chem. Soc. 126:4534 (2004); (e) Dowex 50 H⁺, MeOH;(f) Na, MeOH, 185° C.; (g) 12 N HCl; (h) i) H₂, Rh on Al₂O₃, ii) 0.5 MH₂SO₄, reflux; (i) SO₃, H₂SO₄; (j) NaOH, 350° C.; (k) HZSM-12 zeolite,propene; and (l) i) O₂, ii) H₂O₂, iii) H⁺.

FIG. 3 presents putative reaction pathways according to the presentinvention, by which malonyl-CoA is biosynthetically converted tophloroglucinol by a phloroglucinol synthase, either via enzyme-activated3,5-diketopimelate (3,5-diketoheptanedioate) or via enzyme-activated3,5-diketohexanoate (3,5-diketocaproate).

FIG. 4 illustrates a variety of exemplary pathways for utilization ofdifferent carbon sources in a process for anabolic phloroglucinolsynthesis. Dashed arrows show possible alternative carbon sourceutilization routes; square brackets enclose intermediates that may beabsent in some pathways.

It should be noted that the figures set forth herein are intended toexemplify the general characteristics of an apparatus, materials andmethods among those of this invention, for the purpose of thedescription of such embodiments herein. These figures may not preciselyreflect the characteristics of any given embodiment, and are notnecessarily intended to define or limit specific embodiments within thescope of this invention.

DETAILED DESCRIPTION

The present invention provides methods, materials and organisms forproducing phloroglucinol and derivatives thereof. The present inventionis based on work in which it was surprisingly discovered that, insteadof expressing multiple genes involved in the microbialacetylphloroglucinol pathway, e.g., phlABCDEF, expression of a singlegene could produce substantial concentrations of phloroglucinol itself.Publications, such as U.S. Pat. No. 6,051,383, Thomashow et al., issuedApr. 18, 2000, led to the conclusion that, if phloroglucinol were to beproduced by use of such genes, it was a highly likely possibility thatthe best or only route to do so would be to add at least one more enzymeto the pathway to de-acetylate the acetylphloroglucinols.

Instead, it has now been unexpectedly found that expression of a singleenzyme, named herein as “phloroglucinol synthase” and expressed from aphlD gene, results in formation of phloroglucinol directly, i.e. notthrough an acetylated or diacetylated intermediate. As a result, thisone enzyme can be expressed alone, apart from any other phl operongenes, to obtain significant levels of phloroglucinol synthesis. Inaddition, this route of phloroglucinol synthesis proceeds through a3,5-diketohexanoate thioester intermediate, not through a3,5,7-triketooctanoate thioester. It has been further discovered thatexpression of the phloroglucinol synthase per se, apart from any otherphl operon genes, has now been demonstrated to be capable of producingsignificantly more phloroglucinol than any other biosynthetic orsemi-biosynthetic route proposed to date; and that phloroglucinol isthereby produced in a simpler, more efficient and economic manner thanany other biosynthetic or semi-biosynthetic route yet proposed.

The headings (such as “Introduction” and “Summary,”) and sub-headingsused herein are intended only for general organization of topics withinthe disclosure of the invention, and are not intended to limit thedisclosure of the invention or any aspect thereof. In particular,subject matter disclosed in the “Introduction” may include aspects oftechnology within the scope of the invention, and may not constitute arecitation of prior art. Subject matter disclosed in the “Summary” isnot an exhaustive or complete disclosure of the entire scope of theinvention or any embodiments thereof.

The citation of references herein does not constitute an admission thatthose references are prior art or have any relevance to thepatentability of the invention disclosed herein. Any discussion of thecontent of references cited in the Introduction is intended merely toprovide a general summary of assertions made by the authors of thereferences, and does not constitute an admission as to the accuracy ofthe content of such references. All references cited in the Descriptionsection of this specification are hereby incorporated by reference intheir entirety.

The description and specific examples, while indicating embodiments ofthe invention, are intended for purposes of illustration only and arenot intended to limit the scope of the invention. Moreover, recitationof multiple embodiments having stated features is not intended toexclude other embodiments having additional features, or otherembodiments incorporating different combinations the stated of features.

As used herein, the words “preferred” and “preferably” refer toembodiments of the invention that afford certain benefits, under certaincircumstances. However, other embodiments may also be preferred, underthe same or other circumstances. Furthermore, the recitation of one ormore preferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

As used herein, the word “include,” and its variants, is intended to benon-limiting, such that recitation of items in a list is not to theexclusion of other like items that may also be useful in the materials,compositions, devices, and methods of this invention.

The word “recombinant” is used herein to indicate that nucleic acidmanipulation was employed. As a result, phrases such as “recombinant”nucleic acid, “recombinant” polypeptide, and “recombinant” cell toentities that were produced, at least in part, by nucleic acidmanipulation. The term “in vivo” is used herein to include “in cyto”where the cell is a living cell.

Sequence Homology

In a preferred embodiment, a mutant polypeptide according to the presentinvention will have an amino acid sequence that is at least 50%homologous to that of a native polypeptide performing the same functionas the mutant. By way of example, a phloroglucinol synthase according tothe present invention will have an amino acid sequence at least 50%homologous to that of SEQ ID NO:2; in a preferred embodiment, thesequence will be at least 60% homologous thereto; in a preferredembodiment, the sequence will be at least 70% homologous thereto; in apreferred embodiment, the sequence will be at least 80% homologousthereto; in a preferred embodiment, the sequence will be at least 90%homologous thereto.

In one embodiment, a recombinant polynucleotide according to the presentinvention, which encodes a desired polypeptide, will be any that encodesa polypeptide having homology to a native polypeptide of the samefunction, as described above. In one embodiment, a recombinantpolynucleotide according to the present invention, which encodes adesired polypeptide, will have an amino acid sequence that is more than80% homologous to that of a native polynucleotide encoding a polypeptideperforming the same function as the mutant. In a preferred embodiment,the polynucleotide will be at least 85% homologous thereto; in apreferred embodiment, the polynucleotide will be at least 90% homologousthereto; in a preferred embodiment, the polynucleotide will be at least95% homologous thereto.

Sequence homology refers to the degree of identicality between twosequences of amino acid residues, or between two sequences ofnucleobases. This may be determined by visual comparison of twosequences, or by use of bioinformatic algorithms that align sequencesfor comparison or that determine percent homology among comparedsequences. Useful automated algorithms are available in the GAP,BESTFIT, FASTA, and TFASTA computer software modules of the WisconsinGenetics Software Package (available from Genetics Computer Group,Madison, Wis., USA). The alignment algorithms automated in those modulesinclude the Needleman & Wunsch, the Pearson & Lipman, and the Smith &Waterman sequence alignment algorithms. Other useful algorithms forsequence alignment and homology determination are automated in softwareincluding: FASTP, BLAST, BLAST2, PSIBLAST, and CLUSTAL V; see, e.g., N.P. Brown et al., Bioinformatics: Applications Note, 1998, 14:380-81; theU.S. National Center for Biotechnology Information athttp://www.ncbi.nlm.nih.gov/Tools/index.html; and U.S. Pat. No.6,790,639, Brown et al., issued Sep. 14, 2004, which provides softwareparameter settings useful for homology determination herein.

The sequence homology exhibited by nucleobase polymers, such as nucleicacids and nucleic acid analogs, may be determined by hybridizationassays between a first sequence and the complement of a second sequence.Any of the well known hybridization assays may be used for this purpose,and examples of these include those described in U.S. Pat. No.6,767,744, Koffas et al., issued Jul. 27, 2004, and U.S. Pat. No.6,783,758, Wands et al., issued Aug. 31, 2004, with “high stringency”hybridization conditions being as defined therein.

Conservative Substitutions

In addition, conservative amino acid substitutions may be present in apolypeptide according to the present invention. The term “conservativeamino acid substitution” indicates any amino acid substitution for agiven amino acid residue, where the substitute residue is so chemicallysimilar to that of the given residue that no substantial decrease inpolypeptide function (e.g., enzymatic activity) results. Conservativeamino acid substitutions are commonly known in the art and examplesthereof are described, e.g., in U.S. Pat. No. 6,790,639, Brown et al.,issued Sep. 14, 2004; U.S. Pat. No. 6,774,107, Strittmatter et al.,issued Aug. 10, 2004; U.S. Pat. No. 6,194,167, Browse et al., issuedFeb. 27, 2001; or U.S. Pat. No. 5,350,576, Payne et al, issued Sep. 27,1994. In a preferred embodiment, a conservative amino acid substitutionwill be any one that occurs within one of the following six groups

1. Small aliphatic, substantially non-polar residues: Ala, Gly, Pro,Ser, and Thr;

2. Large aliphatic, non-polar residues: Ile, Leu, and Val; Met;

3. Polar, negatively charged residues and their amides: Asp and Glu;

4. Amides of polar, negatively charged residues: Asn and Gln; His;

5. Polar, positively charged residues: Arg and Lys; His; and

6. Large aromatic residues: Trp and Tyr; Phe.

In a preferred embodiment, a conservative amino acid substitution willbe any one of the following, which are listed as Native Residue(Conservative Substitutions) pairs: Ala (Ser); Arg (Lys); Asn (Gln;His); Asp (Glu); Gln (Asn); Glu (Asp); Gly (Pro); His (Asn; Gln); Ile(Leu; Val); Leu (Ile; Val); Lys (Arg; Gln; Glu); Met (Leu; Ile); Phe(Met; Leu; Tyr); Ser (Thr); Thr (Ser); Trp (Tyr); Tyr (Trp; Phe); andVal (Ile; Leu).

Just as a polypeptide may contain conservative amino acidsubstitution(s), a polynucleotide hereof may contain conservative codonsubstitution(s). A codon substitution is considered conservative if,when expressed, it produces a conservative amino acid substitution, asdescribed above. Degenerate codon substitution, which results in noamino acid substitution, is also useful in polynucleotides according tothe present invention. Thus, e.g., a polynucleotide encoding a selectedpolypeptide useful in an embodiment of the present invention may bemutated by degenerate codon substitution in order to approximate thecodon usage frequency exhibited by an expression host cell to betransformed therewith, or to otherwise improve the expression thereof.

Moreover, nucleobase sequence-containing polymers, such as nucleicacids, will preferably include those encoding an enzyme or enzymesaccording to the present invention. In addition, these will include,e.g., nucleic acids sharing at least 80% sequence homology with a givenenzyme-encoding nucleic acid. By way of example, a phloroglucinolsynthase coding sequence according to the present invention will have abase sequence at least 80% homologous to that of SEQ ID NO:1; in apreferred embodiment, the sequence will be at least 85% homologousthereto; in a preferred embodiment, the sequence will be at least 90%homologous thereto; in a preferred embodiment, the sequence will be atleast 95% homologous thereto.

Production of Phloroglucinol and its Derivatives

As illustrated in FIG. 3, a phloroglucinol synthase has now beenidentified and characterized, and the mechanism by which this enzymecatalyzes phloroglucinol synthesis has been unexpectedly found toproceed according to the following series of steps, or via analternative mechanism in which the first malonyl-CoA providing the groupthat is transferred to form the illustrated thioester (—SR) linkage,provides a malonyl, rather than an acetyl group, therefor.

-   -   Acetyl Activation—The first step involves activation of an        acetyl group. This occurs by decarboxylation of malonyl-CoA to        transfer an acetyl group therefrom to the enzyme, thus forming        an enzyme-activated acetyl thioester (“R” in FIG. 3 represents        the enzyme or a moiety attached thereto); in an alternate        embodiment, the first step involves activation of an entire        malonyl group to form an enzyme-activated malonyl thioester;    -   Chain Extension—The next phase involves two successive        malonyl-CoA decarboxylations to transfer further acetyl groups        to form an enzyme-activated 3-ketobutanoate thioester and then        an enzyme-activated 3,5-diketohexanoate thioester; in an        alternate embodiment, successive transfers form        enzyme-activated: 3-ketoglutarate thioester and        3,5-diketopimelate thioester;    -   Cyclization—The final step involves cyclization of the        3,5-diketohexanoate thioester intermediate to form        phloroglucinol; in an alternative embodiment, a decarboxylation        of 3,5-diketopimelate takes place to permit cyclization to        phloroglucinol.        All three steps are catalyzed by phloroglucinol synthase.

An enzyme system according to the present invention will contain atleast one phloroglucinol synthase. In a preferred embodiment, thephloroglucinol synthase will be obtained from a Pseudomonad; in apreferred embodiment it will be obtained from a member of the genusPseudomonas; in a preferred embodiment, it will be obtained from amember of the species P. fluorescens. In a preferred embodiment, it willbe obtained from P. fluorescens Pf-5. The amino acid sequence of the P.fluorescens Pf-5 phloroglucinol synthase identified in the present workis shown in SEQ ID NO:2, and its native coding sequence is shown in SEQID NO:1.

In a preferred embodiment, an enzyme system according to the presentinvention will further contain at least one enzyme capable, eithersolely or jointly with other enzyme(s), of catalyzing the formation ofmalonyl-CoA. Malonyl-CoA may be biosynthetically produced, e.g., fromacetyl-CoA by a malonyl-CoA synthesis enzyme, such as: the malonyl-CoAsynthetase (MatB) from Rhizobium leguminosarum (see GenBank AccessionAAC83455 [gi:3982573]), which converts malonate to malonyl-CoA; themalonyl-CoA decarboxylase (MatA) from Rhizobium leguminosarum (seeGenBank Accession AAC83456 [gi:3982574]), which converts malonicsemialdehyde to malonyl-CoA; or the transcarboxylase activity ofacetyl-CoA carboxylase (EC 6.4.1.2), which carboxylates acetyl-CoA toform malonyl-CoA. The malonic acid, malonic semialdehyde, or acetyl-CoAstarting material may be, and preferably is, biosynthetic; for example,the acetyl-CoA may have been biosynthetically derived from any one of avariety of sources, such as glucose, photosynthetic 3-phosphoglycerate,etc.

An enzyme system according to the present invention can be either invitro or in vivo. Where a malonyl-CoA synthesis enzyme is not provided,malonyl-CoA will be supplied to the medium in contact with the cellsand/or enzymes. In one embodiment, a phloroglucinol synthase-encodingnucleic acid may be transformed into cells of an organism capable ofsynthesizing malonyl CoA, in which case phloroglucinol may be producedtherein. Examples of organisms synthesizing malonyl CoA include plants,algae, animals, and humans. In vitro systems include, e.g., batch enzymesuspensions or (adsorbed or covalently) immobilized enzyme bioreactors.In vivo systems include, e.g., immobilized cell bioreactors, continuousfermentations, and batch fermentations. “Fermentation” as used hereinindicates cultured cell growth under any effective conditions, ratherthan a requirement for, e.g., anaerobic conditions or anaerobicmetabolism, which is merely permitted as another embodiment hereof. Inany embodiment, a source of malonyl-CoA will be provided to thephloroglucinol synthase, whether or not that source is added (e.g.,exogenous) malonyl-CoA or in situ biosynthesized (e.g., endogenous)malonyl-CoA.

Recombinant cells according to the present invention are capable ofexpressing at least one phloroglucinol synthase and optionally at leastone malonyl-CoA synthesis enzyme, but neither an entire phlABCD operonnor all three of phlA, phlB, and phlC genes. In a preferred embodiment,a recombinant cell will be one that is capable of expressing arecombinant phloroglucinol synthase therein. In a preferred embodiment,the recombinant cell capable of expressing phloroglucinol synthase andoptionally of expressing a malonyl-CoA synthesis enzyme, will be awalled cell. Examples of walled cells include plant cells, yeast/fungalcells, bacterial cells, Archaea cells, and some protists. In oneembodiment, the recombinant cell will be an avascular plant (e.g.,moss), protist (e.g., algae), yeast, fungal, bacterial, or archaealcell. In one embodiment, the recombinant cell will be a recombinantmicrobe. In one embodiment, the recombinant cell will be a yeast,fungal, bacterial, or archaeal cell, more preferably a yeast, fungal, orbacterial cell. In a preferred embodiment, the recombinant cell will bea bacterial cell. In a preferred embodiment, the recombinant cell willbe a proteobacterial cell. Preferably, the recombinant cell will lackthe ability to express functional enzymes from phlABC, phlE, and phlFgenes. In a preferred embodiment, the cell will be a phlABC⁻, phlE⁻, andphlF⁻ cell. Recombinant host cells will contain at least one nucleicacid encoding a phloroglucinol synthase according to the presentinvention. In a preferred embodiment, the nucleic acid will be in theform of a vector, such as a plasmid or transposon.

In one embodiment for forming a useful phlD⁺ recombinant cell hereof, acell that is both phlD⁺ and phlA⁺, phlB⁺, and/or phlC⁺ will be madephlA⁻, phlB⁻, and/or phlC⁻, as by any gene knockout technique (i.e. anygene excision or mutation technique that results in the cell's inabilityto make the functioning expression product encoded by thepre-knocked-out gene). Preferably, all of the phlA, phlB, and phlC genespresent in the cell will be knocked out. The resulting cell will retainits phlD⁺ phenotype. Optionally, phlE and/or phlF genes present in thecell may also be knocked out. In one preferred embodiment, a phlABCD⁺cell will be made phlABC⁻. In one embodiment, a cell that is both phlD⁻and phlA⁻, phlB⁻, and/or phlC⁻ will be made phlD⁺ by inserting anexpressible PhlD-encoding nucleic acid into the cell, whether into thegenomic DNA thereof or as part of an extrachromosomal unit, such as aplasmid, or both. In one preferred embodiment, a phlABCD⁻ cell will bemade phlD⁺.

In some embodiments, a native or recombinant cell that is PhlD⁺, such asa phlD⁺ cell, can be further supplemented with additional phlD gene(s),as by transformation with nucleic acid comprising one or moreexpressible open reading frames encoding a phloroglucinol synthase. ThePhlD⁺ cell may be a PhlA⁻, PhlB⁻, and/or PhlC⁻ cell, such as a phlA⁻,phlB⁻, and/or phlC⁻, or it may be a PhlA⁺, PhlB⁺, and/or PhlC⁺ cell,such as a phlA⁺, phlB⁺, and/or phlC⁺ cell (e.g., a phlABCD⁺ cell). Theresulting recombinant cell, which is capable of expressing theadditional phlD gene(s) can exhibit enhanced phloroglucinol synthesiscapability.

Similarly to recombinant cells, isolated or recombinant enzyme systemsaccording to the present invention comprise at least one phloroglucinolsynthase, and optionally at least one malonyl-CoA synthesis enzyme orenzyme set, but do not comprise all three of PhlA, PhlB, and PhlCenzymes, and preferably comprise none of PhlA, PhlB, and PhlC enzymes.Thus, recombinant cells and enzyme systems according to the presentinvention, which comprise at least one phloroglucinol synthase andoptionally at least one malonyl-CoA synthesis enzyme, but do comprisefewer than all three of PhlA, PhlB, and PhlC enzymes, can both bereferred to as PhlD⁺ entities that are at least one of PhlA⁻, PhlB⁻, andPhlC⁻, and preferably are PhlABC⁻.

Processes for the production of phloroglucinol involve contacting aphloroglucinol synthase with malonyl-CoA. Processes for the productionof resorcinol involve performing a hydrogenation reaction uponbiosynthetic phloroglucinol, e.g., using hydrogen and a rhodiumcatalyst. Phloroglucinol and resorcinol produced by a process accordingto the present invention may be used in or as, or to prepare,compositions such as medicaments, cosmetics, dyes, polymer resins,rubbers, adhesives, sealants, coatings, propellants, explosives,composite materials, and laminated or bonded materials.

Whole-Cell Fermentation Modes

Whole cell fermentations of recombinant cells hereof may be performed inany culture mode, preferably in a batch, fed-batch, or continuous (orsemi-continuous, i.e. reseeding) mode. In some embodiments hereof,phloroglucinol-containing spent medium produced by a culture ofrecombinant cells according to the present invention is processed toextract phloroglucinol. However, in some cases, phloroglucinol itself,when it reaches a threshold concentration in the growth medium, canexert toxicity against the cultured cells in a process calledend-product inhibition. Where present, this inhibition decreasescellular production of phloroglucinol and can result in reduced cellviability. Thus, in some embodiments, an extraction of phloroglucinol,and optionally phloroglucinol derivatives (if present), from anotherwise still-useful growth medium is preferably performed during theperiod in which the cells are actively producing phloroglucinol. Such anembodiment is referred to herein as an “extractive fermentation.”

Extractive fermentation can be performed herein in any of the modesknown useful in the art. For example, some embodiments employ adispersed extractive fermentation mode in which an extractive absorbentor adsorbent liquid or particle phase, which is capable of uptakingphloroglucinol, is introduced into the medium in which the recombinantcells are grown, where the extractive particles or liquid zones comeinto contact with and uptake phloroglucinol. As in any extractiveprocess, uptake of this product by the absorbent or adsorbent materialcan be non-specific, preferential, or specific, and is preferablypreferential or specific for phloroglucinol.

After becoming “loaded” with phloroglucinol, the “loaded” extractivephase is removed from the culture medium, e.g., by centrifugation,filtration, magnetic collection of magnetic or magnetizable particles,and/or by phase separation such as where the extractive phase risesabove or sinks below the bulk of the culture medium. In someembodiments, a counter-current or cross-current extraction technique maybe utilized to extract phloroglucinol from the culture medium, such aswhere the stream that is counter-current or cross-current to the culturemedium stream comprises such an extractive liquid or particle phase.

In some embodiments, a membrane extractive fermentation is performed bypassing the culture medium over an extraction membrane, such as an ionexchange membrane. In some embodiments, a column extractive fermentationis performed by passing the culture medium through an extraction column,such as a hollow fiber membrane extractor or a fibrous or bead resincolumn. The cells in culture in the medium may pass through the column,or some or all of the cells may be removed, e.g., by filtration, beforethe medium is passed through the column. In any extractive fermentationprocess, the extractive fermentation can be performed once, multipletimes, or continuously during the fermentation process. Incounter-current, cross-current, membrane, and column extractivefermentation modes, the medium from which some or all (preferably mostor all) of the phloroglucinol has been removed, is then returned to thefermentation vessel.

In a preferred embodiment, a column extractive fermentation technique isemployed to remove phloroglucinol from the culture medium duringbiosynthesis. Useful media for this purpose include anion exchangemedia, such as anion exchange beads, fibers, and hollow fibers. Anionexchange membranes and anion exchange media particles are likewiseuseful in membrane and dispersed extractive fermentation modes,respectively. Where a particulate anion exchange medium is used, it willpreferably be utilized in a fluidized bed extractive fermentation mode,although stationary bed modes may alternatively be used.

Useful anion exchange media may comprise any support, whether organic orinorganic, that contains or is covalently attached to anion exchangegroups. In some embodiments, an organic support will be used, such as astyrene-divinylbenzene, polystyrene, polyvinyl, acrylic,phenol-formaldehyde, organosilicon, or cellulose polymer backbone,wherein the backbone comprises or is attached to anion exchange groups.

Useful anion exchange groups may be any cationic group, preferablynon-metal cationic groups, such as organic ammonium, sulfonium, andphosphonium groups. Preferred cationic groups include organic: tertiaryammonium (e.g., diethylaminoethyl cellulose), quaternary ammonium,pyridinium, tertiary sulfonium, and quaternary phosphonium groups. Inone embodiment, the anion exchange groups of the anion exchange mediumwill be quaternary ammonium or pyridinium groups. Examples of quaternaryammonium-type resins include AG-1 X8 resin (from Bio-Rad LaboratoriesInc., Hercules, Calif., USA) and DOWEX 1 resin (from The Dow ChemicalCo., Midland, Mich., USA); examples of pyridinium-type resins includepolyvinyl-alkyl-pyridinium resins obtainable by alkyl halide treatmentof polyvinyl-pyridine resins, such as REILLEX HP (from ReillyIndustries, Inc., Indianapolis, Ind., USA), or obtained directly fromcommercial sources, such as poly(4-vinyl N-methyl pyridinium iodide)(from Polymer Source Inc., Montreal, QC, CA).

In one preferred embodiment, the anion exchange medium will be treatedto prepare a phosphate complex with cationic groups of the medium, priorto use. Where an anion exchange medium is re-used (without interveningremoval of phloroglucinol therefrom), or is in continuous contact, witha given fermentation, it will preferably be replaced with new or renewedanion exchange medium frequently enough that the phloroglucinolconcentration of the culture medium does not rise to a level at which asubstantial degree of end-product-inhibition would occur; preferably notabove about 2 g/L, or not above about 1.5 g/L, phloroglucinol.

Anion exchange medium that has already been loaded with phloroglucinol,by either an extractive fermentation or a post-fermentation extractionprocess, is preferably treated to removal phloroglucinol by washing itwith water, acidified water, acidified alcohol (e.g., acidic ethanol) ora combination thereof. Water washing followed by acidified alcoholwashing is one preferred technique. After (preferably) most or all ofthe phloroglucinol has been removed from the anion exchange medium, thatmedium can be prepared for re-use in phloroglucinol extraction, e.g., byequilibrating it with a phosphate solution to form cationicgroup-phosphate complexes prior to re-use. Phloroglucinol present in thewashes may be further isolated and/or purified by any techniques knownin the art, e.g., phase separation, solvent evaporation, and so forth.

Whole-Cell Fermentation Conditions

A culture of whole cells used in a method for producing phloroglucinolaccording to the present invention will utilize conditions that arepermissive for cell growth and those that permit the cultured cells toproduce anabolic phloroglucinol. In some cases, a phloroglucinolsynthase will be expressed throughout the cell culture period, e.g.,constitutively; yet, in many cases, it is desirable to begin expressingphloroglucinol synthase only near the end of the exponential growthphase (EGP). Where a later expression is desired, a phloroglucinolsynthase coding sequence that is under the control of a regulatedpromoter generally will be activated or derepressed when about 70 to100%, preferably when about 70 to about 90%, more preferably when about70 to about 80% of EGP has elapsed. Examples of promoters useful forthis purpose include the tac, T5, and T7 promoters; P_(T7) is preferredamong these; induction may be made using lactose or a gratuitous inducersuch as IPTG (isopropyl-beta-D-thiogalactopyranoside).

In some preferred embodiments hereof, a recombinant microbial cell, suchas a recombinant bacterial host cell will be used as a whole cellbiocatalyst herein. Preferred bacteria for this purpose includeproteobacteria; preferred examples of proteobacteria include the gammaproteobacteria, such as enterobacteria and pseudomonads; Escherichiaspp., such as E. coli, and Pseudomonas spp., such as P. fluorescens, arepreferred among these. Preferred microbes are those that lack or havebeen treated to decrease or eliminate protease activities that would becapable of degrading the phloroglucinol synthase and/or malonyl-CoAsynthesis enzymes according to the present invention. In bacteria, Lonand OmpT are two proteases that are preferably absent or are otherwisedecrease or eliminated, e.g., by mutation. E. coli strains BL21 andW3110 are preferred examples of phlABCD⁻ cells for insertion of phlDgene(s); and P. fluorescens strain Pf-5 is a preferred example ofphlABCD⁺ cells for inactivation of phlA, phlB, and/or phlC, with orwithout insertion of further phlD gene(s), or for inactivation ofphlABCD, with insertion of further phlD gene(s), or for supplementationwith additional phlD gene(s). E. coli strain BL21 may be obtained as:BL21 STAR (DE3) ONE SHOT (Invitrogen Corp., Carlsbad, Calif., USA); orULTRA BL21 (DE3) (Edge BioSystems, Gaithersburg, Md., USA). E. colistrain W3110 may be obtained as ATCC No. 27325 (American Type CultureCollection, Manassas, Va., USA); and P. fluorescens strain Pf-5 may beobtained as ATCC No. BAA-477.

In the case of E. coli, preferred fermentation temperatures are fromabout 20 to about 37° C., preferably about 25 to about 37° C., morepreferably about 30 to about 37° C. It has been discovered that, in thecase of anabolic phloroglucinol synthesis, a combination of a highertemperature during EGP, or at least during the pre-induction portion ofEGP, and a lower temperature during at least part of the remainingculture period (e.g., throughout all or part of the post-induction orall or part of the maintenance phase), is an important feature of asuperior phloroglucinol production protocol. Thus, in one preferredembodiment, recombinant E. coli cells will be grown at about 35-37° C.,preferably at about 36-37° C., more preferably at about 36° C. duringEGP, or during pre-induced EGP; and at about 30-34° C., preferably atabout 30 to about 33° C., more preferably at about 33° C. or about 30°C. during maintenance phase, or during post-induction. In someembodiments, the switch to a lower temperature may occur well into themaintenance phase, e.g., up to about 15 hours after EGP has ended. Thus,in the case of a cell (e.g., E. coli) culture in which EGR ends at about15 hours from the start of culturing, the switch from a higher to alower temperature for a two-temperature fermentation profile may occur,e.g., at about 11 or about 12 hours (e.g., at approximately the sametime as a 70% or 80% EGR induction point), or at about 15 hours, or evenup to about 30 hours from the start of culturing. Alternatively, thehigher temperatures useful, e.g., for EGR in such a dual-temperatureembodiment are useful temperatures for the entire culturing period. Inthe case of P. fluorescens, preferred temperatures are from about 20 toabout 30° C., with preferred higher temperatures being from about 27 toabout 30° C., and preferred lower temperatures being from about 24 toabout 27° C.

Carbon Sources

Cells, enzyme systems, and methods for producing phloroglucinolaccording to embodiments of the present invention will utilize a carbonsource. Where a carbon source other than malonyl-CoA is contacted withthe cell or enzyme system, an enzyme system or recombinant cellaccording to the present invention can utilize any other carbon source,provided that the enzyme system or recombinant cell according to thepracticed embodiment can metabolize the carbon source into substancesthat can be used to anabolically synthesize phloroglucinol as describedherein. The nature of the other enzymology, besides the phloroglucinolsynthase, and the optional malonyl-CoA synthesis activity, present inthe reaction mixture will determine which carbon sources may be used.FIG. 4 illustrates a number of representative routes for anabolicsynthesis of phloroglucinol from carbon sources.

Thus, in the case of most carbon sources, the cell or enzyme system willfirst either catabolize the carbon source or will fix it, in order toprovide simple organic molecules from which the cell may formacetyl-CoA. Alternatively, the carbon source will be directly convertedto acetyl-CoA. For example, in the case of an acetate carbon source, theacetate can be directly converted to acetyl-CoA, or it may first beconverted to acetyl phosphate. Acetyl-CoA may itself be used as a carbonsource. Once obtained or provided, the acetyl-CoA can then be used inthe synthesis of malonyl-CoA, as by action of acetyl-CoA carboxylase.Where degradation of a carbon source produces catabolic malonate orcatabolic malonic semialdehyde, or where malonate or malonicsemialdehyde is present in the carbon source, these may be converted tomalonyl-CoA by a malonyl-CoA synthetase or malonyl-CoA decarboxylase,respectively, present in the enzyme pool of the cell or enzyme system,or they may be catabolized for use in acetyl-CoA synthesis, withsubsequent conversion to malonyl-CoA. Once obtained or provided,malonyl-CoA is then used as a substrate by phloroglucinol synthase,forming phloroglucinol

Where the carbon source comprises a biomolecule-type carbon compound,the carbon compound will preferably be a primary metabolite-typecompound. Examples of primary metabolite-type compounds include any ofthe, preferably C1-C18: fatty acids, waxes, mono-, di-, andtri-glycerides; polyols; aliphatic hydroxy acids; phospholipids;phosphoacids; monosaccharides (e.g., trioses, tetroses, pentoses,hexoses, heptoses, and the like); amino acids; and nucleotides; andhydrolysable homo- and hetero-oligomers (i.e., including -dimers) and-polymers formed from such compounds; and biologically activated formsof such compounds (e.g., acetyl-CoA). Biomolecule-type compounds may beof any origin, whether biological or synthetic. Other preferredcompounds include any small or non-complex organic compounds, i.e.generally C1-C18, aliphatic cycloaliphatic, and aromatic compounds, andthe like, of any source, having a preferred monomeric complexity of 4 orfewer carbon-carbon branch points per 18 carbon atoms, e.g.: C1-C18aliphatic hydrocarbons and their mono- and poly-acids, -alcohols,-amines, -carbonyls; and hydrolysable homo- and hetero-oligomers and-polymers formed therefrom.

Carbon sources comprising such small/non-complex organic(s), and/orprimary metabolite-type compound(s), without substantial concentrationsof (and preferably less than 10%-of-carbon-by-weight concentrations of)secondary metabolites or of larger-monomer-type or complex organiccompounds are also referred to herein as “simple” carbon sources. Asused herein, secondary metabolites include, e.g.: alkaloids; coumarins;polyketides; terpenoids, isoprenoids, sterols, steroids, andprostaglandins; catecholamines; porphyrins; xanthones; flavonoids;phenylpropanoids and phenolics (including, e.g., benzenoids andpolyphenolics); and the like. Large or complex organic compounds arealiphatic, cycloaliphatic, and aromatic compounds, and the like, thathave a monomeric compound size above C18 and/or a monomeric compoundcomplexity above 4 carbon-carbon branch points per 18 carbon atoms.

In a preferred embodiment, a carbon source will be a simple carbonsource. Preferred simple carbon sources contain from 0% to about 5%,more preferably from 0% to about 2%, or 0% to about 1%, or 0% to about0.5%, or preferably about 0% by weight secondary metabolites and largeror complex organics; or preferably free or at least substantially freeof secondary metabolites and larger/complex organics. In someembodiments, a simple carbon source will comprise primarymetabolite-type compound(s). Preferred examples of primarymetabolite-type compound(s) for use herein include: saccharides,preferably mono- and/or di-saccharides; and polyols. Glucose, xylose,and arabinose are preferred examples of a monosaccharide for use in acarbon source herein; glycerol is one preferred example of a polyoltherefore. In one embodiment hereof, glucose, xylose, and/or arabinosewill be used as the carbon source, preferably as the carbon sourcethroughout both the exponential growth phase and the maintenance phaseof the cell culture. In one embodiment a combination of amonosaccharide(s) (preferably glucose, xylose, and/or arabinose) andglycerol will be used, e.g., a 1:1 or 2:1 weight ratio; preferably, sucha combination will be used only during the maintenance phase, with amonosaccharide(s) (without glycerol) being used during the exponentialgrowth phase.

EXAMPLES

Phloroglucinol (1a, Scheme 1, i.e. FIG. 1) is found as a substituent ina variety of natural products. However, biosynthesis of phloroglucinol1a as a free-standing molecule has not been delineated. As part of asearch for such biosynthetic activity, formation of acetylatedphloroglucinols (6 and 7, Scheme 1) in Pseudomonas fluorescens Pf-5 isexamined. B. Novak-Thompson et al., Can. J. Microbiol. 40:1064 (1994).Phloroglucinol biosynthesis is detected. Subsequent heterologousexpression of P. fluorescens phlD leads to accumulation ofphloroglucinol 1a in Escherichia coli cultures. Beyond the implicationsrelevant to the biosynthesis of acetylphloroglucinols, these discoveriesestablish the basis for new, environmentally-benign syntheses ofphloroglucinol and resorcinol.

Activated 3,5-diketohexanoate 2 (i.e. activated 3,5-diketocaproate)would be a likely precursor to phloroglucinol 1a and triacetic acidlactone 3a (Scheme 1), which are two of the structurally simplestpolyketide natural products. For example, 2-pyrone synthase from Gerberahybrida catalyzes the conversion of malonyl-CoA into triacetic acidlactone. S. Eckermann et al., Nature 396:387 (1998); J. Jez et al.,Chem. Bio. 7:919 (2000). Alteration of an active site tyrosine to aphenylalanine results in formation of triacetic acid lactone as theexclusive biosynthetic product in Brevibacterium ammoniagenes fatty acidsynthase B. W. Zha et al., J. Am. Chem. Soc. 126:4534 (2004).Phloroglucinol formation is not observed with any of thesenaturally-occurring or mutant enzymes. Another, alternative possibleprecursor to phloroglucinol 1a is activated 3,5-diketo-n-heptanedioate12. (i.e. activated 3,5-diketopimelate).

Prospecting for the biosynthesis of phloroglucinol 1a leads to P.fluorescens Pf-5 and biosynthesis of 2,4-diacetylphloroglucinol 7(Scheme 1). B. Novak-Thompson et al., Can. J. Microbiol. 40:1064 (1994).Acetylphloroglucinol biosynthesis is encoded by a gene clusterconsisting of phlACBD, a protein for product export encoded by phlE, anda divergently transcribed phlF-encoded regulator. PhlD been suggested tobe involved in the formation and cyclization of an activated3,5,7-triketooctanoate 5 (Scheme 2). The resulting intermediate2-acetylphloroglucinol 6 is then presumably acetylated to from2,4-diacetylphloroglucinol 7 (Scheme 2). See M. G. Bangera & L. S.Thomashow, J. Bacteriol. 181:3155 (1999). Biosynthesis of phloroglucinol1a is not an activity that has been assigned to PhlD.

P. fluorescens Pf-5/pME6031 is examined for products that accumulate inits culture supernatants. In addition (entry 1, Table 1) to accumulationof 2,4-diacetylphloroglucinol 7 and 2-acetylphloroglucinol 6, formationof phloroglucinol is discovered. To increase the concentration ofbiosynthesized phloroglucinols, P. fluorescens PF-5 is transformed withpJA2.232, a plasmid derived from the insertion of the phlABCDE genecluster into pME6031. The goal is to evade regulation bygenomically-encoded PhlF by presenting multiple copies of thebiosynthetic gene cluster. This approach results in large increases inthe concentrations of phloroglucinols 1a, 6, 7 synthesized by P.fluorescens Pf-5/pJA2.232 (entry 2, Table 1) relative to P. fluorescensPf-5/pME6031 (entry 1, Table 1). TABLE 1 Maximum Concentrations ofPhloroglucinol 1a, 2-Acetylphloroglucinol 6, and2,6-Diacetylphlorogluinol 7 Biosynthesized by Constructs ExpressingphlACBDE Genes. Host/ plasmid phloroglucinols (mg/L) entry plasmidinserts 1a 6 7 1 P. fluorescens Pf-5/ (none) 10 23 35 pME6031^(a) 2 P.fluorescens Pf-5/ phlACBDE 470 500 790 pJA2.232^(a) 3 E. coli BL21(DE3)/phlACBDE 32 14 0 pJA3.085^(b) 4 E. coli BL21(DE3)/ phlACBD 22 13 0pJA3.156^(b) 5 E. coli BL21(DE3)/ phlD 720 0 0 pJA2.042^(b) 6 E. coliJWF1(DE3)/ phlD 780 0 0 pJA3.131A^(c) 7 E. coli BL21(DE3)/ phlACB 0 0 0pJA3.169^(b) 8 E. coli BL21(DE3)/ phlACB 39 17 2 pJA3.169^(b,d)^(a)Cells are cultured in YM medium under shake-flask conditions.^(b)Cells are cultured under shake-flask conditions in TB medium andharvested. Following resuspension in M9 minimal salts medium, cells arecultured under shake-flask conditions.^(c)Cells are cultured in M9 minimal salts medium underfermentor-controlled conditions.^(d)Phloroglucinol (50 mg/L) added after cells are resuspended in M9minimal salts.

Further analysis follows from heterologous expression from a T7 promoterof phlACBDE genes in Escherichia coli (entries 3-8, Table 1). All E.coli constructs also carry a chromosomal gene1 insert encoding the T7RNA polymerase. E. coli B121(DE2)/pJA3.085, which carries a phlACBDEplasmid insert, synthesizes phloroglucinol and 2-acetylphloroglucinolbut no 2,4-diacetylphloroglucinol (entry 3, Table 1). The absence of thephlE-encoded product exporter in E. coli B121(DE3)/pJA3.156 does nothave an adverse impact on the concentrations of biosynthesizedphloroglucinol 1a and 2-acetylphloroglucinol 2 (entry 4, Table 1).Product formation attendant with heterologous expression of only phlD isthen evaluated using E. coli BL21(DE3)/pJA2.042 (entry 5, Table 1). Onlyphloroglucinol 1a formation is observed. The differences in theconcentrations of phloroglucinol 1a biosynthesized by E. coliBL21(DE3)/pJA2.042 relative to E. coli B121(DE3)/pJA3.156 (entry 4 vs.entry 5, Table 1) likely reflect the proximity of phlD to the T7promoter. Synthesis of phloroglucinol from glucose in minimal saltsmedium under fermentor-controlled conditions is examined using E. coliJWF1(DE3)/pJA3.131A (entry 6, Table 1).

To further explore its role in phloroglucinol biosynthesis, PhlD ispurified to homogeneity and its in vitro enzymology examined. Noactivity (Table 1) is observed when acetyl-CoA alone is employed as asubstrate. Approximately equal specific activities are observed whenmalonyl-CoA and acetyl-CoA are incubated with PhlD relative toincubation of PhlD with only malonyl-CoA. A K_(m)=37 μM for malonyl-CoAand a k_(cat)=4.7 min⁻¹ are determined for PhlD. For comparison,2-pyrone synthase, which like PhlD employs an activated3,5-diketohexanoate 2 (Scheme 1), is purified to homogeneity. 2-Pyronesynthase is unable to use acetyl-CoA as a substrate. However, incubationof 2-pyrone synthase with malonyl-CoA and acetyl-CoA yields twofoldhigher specific activities relative to incubation with malonyl-CoA inthe absence of acetyl-CoA. Kinetic parameters for 2-pyrone synthaseinclude K_(m)=XX μM for malonyl-CoA, K_(m)=2.2 μM for acetyl-CoA, and ak_(cat)=3.3 min⁻¹.

The products that form upon heterologous expression of PhlACBDE and PhlDraises the possibility that cyclization of an activated3,5-diketohexanoate 2 (Scheme 1) and subsequent stepwise acetylation ofphloroglucinol 1a might be the basis for biosynthesis of2-acetylphloroglucinol 6 and 2,4-diacetylphloroglucinol 7. To furtherexplore this possibility, E. coli BL21(DE3)/pJA3.169 is constructed withplasmid-localized phlACB. M. G. Bangera & L. S. Thomashow, J. Bacteriol.181:3155 (1999). No phloroglucinols are synthesized upon culturing(entry 7, Table 8) of this construct, which lacks plasmid-localizedphlD. However, addition of phloroglucinol 1a to the culture medium of E.coli BL21(DE3)/pJA3.169 does result in formation of2-acetylphloroglucionol 6 and small amounts of2,4-diacetylphloroglucinol 7 (entry 8, Table 1).

PhlD is also of particular importance in establishing the outline of anew synthesis of phloroglucinol, which is currently synthesized (Scheme2, i.e. FIG. 2) from 2,4,6-trinitrotoluene 8 by a route involving anoxidation utilizing Na₂Cr₂O₇. G. Leston, In Kirk-Othmer Encyclopedia ofChemical Technology, Vol. 19, p 778 (J. I. Kroschwitz & M. Howe-Grant,eds.) (4th ed., 1996) (Wiley: New York). Beyond the explosion hazard,environmentally problematic chromates are generated as waste streamsduring synthesis of phloroglucinol 1a from 2,4,6-trinitrotoluene 8.Recently, an alternate route (Scheme 2) to phloroglucinol 1a has beenelaborated involving microbe-catalyzed synthesis of triacetic acidlactone 3a. W. Zha et al., J. Am. Chem. Soc. 126:4534 (2004). Multiplechemical steps are needed to convert triacetic acid lactone 3a intophloroglucinol 1a. C. A Hansen & J. W. Frost, J. Am. Chem. Soc. 124:5926(2002). In contrast to these chemical and chemoenzymatic routes tophloroglucinol, heterologous expression of PhlD in E. coli allowsphloroglucinol 1a to be made in a single microbe-catalyzed step fromglucose (Scheme 2).

An alternative synthesis of resorcinol 9 is also now possible.Resorcinol is currently manufactured (Scheme 2) by alkali fusion of1,3-benzenedisulfonic acid 10 or hydroperoxidation of1,3-diisopropylbenzene 11. Alkali fusion requires high temperatures andgenerates large salt waste streams. Acetone hydroperoxide formed duringhydroperoxidation is an explosion hazard. See L. Krumenacker et al., InKirk-Othmer Encyclopedia of Chemical Technology, Vol. 13, p 996 (J. I.Kroschwitz & M. Howe-Grant, eds.) (4th ed., 1995) (Wiley: New York). Inaddition, both 1,3-benzenedisulfonic acid 10 and 1,3-diisopropylbenzene11 are produced from petroleum-derived, carcinogenic benzene (Scheme 2).The new route to resorcinol 9 is based on the microbial synthesis ofphloroglucinol 1a followed by Rh-catalyzed hydrogenation (Scheme 2) ofthis intermediate. C. A Hansen & J. W. Frost, J. Am. Chem. Soc. 124:5926(2002). Since phloroglucinol 1a can now be synthesized from glucose,resorcinol joins catechol and hydroquinone as dihydroxy aromatics thatare amenable to synthesis from nontoxic, plant-derived glucose (Scheme2). See, respectively: K. D. Draths & J. W. Frost, J. Am. Chem. Soc.117:2395 (1995); and N. Ran et al., J. Am. Chem. Soc. 123:10927 (2001).

Example 2 Expression of PhlD in E. coli Strains and ResultingPhloroglucinol Synthesis

Plasmid pJA3.131A (Kan^(R), lacI^(Q), P_(T7)-phlD, serA) is transfectedinto chromosomally serA⁻ E. coli strains BL21(DE3), W3110(DE3), andJWF1(DE3) [i.e. RB791serA⁻(DE3)], and into strain KL3(DE3) [i.e.AB2834(serA::aroB)] (E. coli strains RB791 and AB2834 are available fromthe E. coli Genetic Stock Center, New Haven, Conn., USA). All DE3strains are obtained by integration of λDE3 prophage into the cellchromosomes. Cells are cultured in fed-batch conditions under mineralsalts and limited glucose. Although all transformed strains expresssubstantial levels of phloroglucinol, the BL21 and W3110 strains producesuperior titers of 3.0 and 3.1 g/L phloroglucinol, respectively; and,relative to the amounts of glucose supplied to the cultures, thesestrains produce a superior phloroglucinol yields of 4.4 and 3.1 molesphloroglucinol per 100 moles of glucose (% mol/mol).

These tests also compare phloroglucinol expression levels in BL21strains similarly transformed with a plasmid in which phlD is under thecontrol of Ptac or P_(T5); P_(T7) is found to provide superior results(data not shown). In these tests, phloroglucinol accumulation for allstrains stops increasing during the stationary (or maintenance) phase.For BL21 and W3110, the highest phloroglucinol concentration is achievedabout 6 hours and about 12 hours, respectively, after initiation ofinduction, i.e. the first IPTG addition. End-product inhibition is alsoobserved. Further tests demonstrate that phloroglucinol is responsiblefor the inhibition when at or above about 2 g/L concentration (data notshown).

Example 3 Extractive Phloroglucinol Fermentation

An anion-exchange resin column-based extractive fermentation is employedto remove phloroglucinol in order to reduce or eliminate itscytotoxicity and phloroglucinol synthesis repression duringfermentation. A stirred tank reactor is equipped with tubing leadingthrough an anion exchange column and returning to the tank; the tubingis equipped with a peristaltic pump in order to circulate the mediumthrough the column. Bio-Rad Econo columns (25×200 mm) packed with 80 mL(bed volume) AG 1-X8 resin are rinsed with 15 bed volumes of KH₂PO₄ (0.8M) to change the tertiary ammonium salts to phosphate form before the insitu extraction. A total of 3 to 5 columns are used for eachfermentation; each column is used for about 6-12 h before being replacedwith another column, in order to keep the culture medium'sphloroglucinol concentration below about 1.5 g/L. All columns areoperated in a fluidized-bed mode and the circulation flow rate is about8-12 mL/min.

To recover the phloroglucinol adsorbed on the AG 1-X8 resin, the columnis washed in a fluidized-bed mode with 10 bed volumes of distilled,deionized water to remove residual cells; this also recovers about 15%of the phloroglucinol from the resin, in the water solution. Then, thecolumn is rinsed in a fixed-bed mode with 15 bed volumes of acidicethanol (acetic acid, 10% (v/v); ethanol, 75% (v/v); H₂O, 15% (v/v)) torecover remaining phloroglucinol from the resin, in the acidic ethanolsolution. After phloroglucinol recovery, the column can be regeneratedby further rinses of 15 bed volumes of KH₂PO₄ (0.8 M), 2 bed volumes ofethanol (70%), and 5 bed volumes of sterilized distilled, deionizedwater, respectively.

To purify the recovered phloroglucinol, cells in the resulting watersolution are removed by centrifugation; the solution is thenconcentrated to about 1/10 of the original volume. Separately, theacidic ethanol solution is concentrated to dryness. This residue isredissolved with the concentrated water solution. The resulting aqueousphase is then extracted three times with an equal volume of ethylacetate. The organic phases are combined, dried over MgSO₄, mixed withsilicone gel, concentrated to dryness, and loaded onto a flash column.Phloroglucinol is separated form other brown impurities by rinsing withhexane:acetate (1:1) and identified by TCL. The fraction containingphloroglucinol is then concentrated to dryness and dried under highvacuum conditions to afford phloroglucinol as pale crystals.

Example 4 Optimization of Phloroglucinol Fermentation

A variety of dual temperature fermentation profiles are used inextractive and non-extractive fermentations of the transformed W3110strain described above. Glucose is steadily fed by pO₂ cascade controland the exhausted CO₂ level is maintained at a steady level until theend of the fermentation. In both types of fermentations, lowering theinitial 36° C. temperature, during fermentation, is found tosignificantly increase the titer and yield of phloroglucinol, withextractive fermentation results being much greater. Temperature shiftsto 30° C. are performed in separate fermentations at 12 h (the time offirst induction by IPTG), 15 h (the beginning of maintenance phase), or30 h. Superior results are obtained when the temperature shift occurs at15 h and the extractive fermentation is permitted to proceed for a totalof 60 h. Under these conditions, the W3110serA⁻(DE3)/pJA3.131Asynthesizes 15 g/L phloroglucinol in a yield of 11% (mol/mol). Incomparison with the non-extractive fermentation, the extractivefermentation is found to provide undiminished phloroglucinol productionthroughout the fermentation, a steady PhlD specific activity, maintainedcell viability, and longer maximum fermentation times.

An identical fermentation profile, with identical extractivefermentation conditions, is also used to test phloroglucinol productionby the BL21serA⁻(DE3)/pJA3.131A strain described above. Equivalentresults to those of the W3110 fermentation are obtained. One furtherdual temperature profile, in which the initial 36° C. temperature isshifted at 15 h to 33° C., is found to increase recovery ofphloroglucinol from BL21 yet further, giving a 17.3 g/L titer and a12.3% (mol/mol) yield.

In addition, expression of recombinant phlD in yeast, S. cerevisiae, issuccessful, although yields are from 0.5 to about 1.5 mg/L under testconditions (data not shown).

The examples and other embodiments described herein are exemplary andnot intended to be limiting in describing the full scope of compositionsand methods of this invention. Equivalent changes, modifications andvariations of specific embodiments, materials, compositions and methodsmay be made within the scope of the present invention, withsubstantially similar results.

1. An isolated or recombinant PhlD⁺ enzyme system that is PhlA⁻, PhlB⁻,and PhlC⁻, or a PhlD⁺ recombinant cell that is PhlA⁻, PhlB⁻, and PhlC⁻,or a PhlD⁺, PhlA⁻, PhlB⁻, and PhlC⁻ recombinant cell that is a PhlD⁺cell that has been genetically engineered to further increase theexpression of PhlD therein, the enzyme system or recombinant cell beingcapable of converting malonyl-CoA to phloroglucinol.
 2. The enzymesystem according to claim 1, wherein said system further comprises atleast one malonyl-CoA synthesis enzyme.
 3. The enzyme system accordingto claim 2, wherein the malonyl-CoA synthesis enzyme is any one ofmalonyl-CoA synthetase, malonyl-CoA decarboxylase, or acetyl-CoAcarboxylase.
 4. The recombinant cell according to claim 1, wherein therecombinant cell is a microbe.
 5. The recombinant cell according toclaim 4, wherein the recombinant cell is a yeast, fungus, or bacterium.6. The recombinant cell according to claim 5, wherein the recombinantcell is a bacterium.
 7. The recombinant cell according to claim 6,wherein the recombinant cell is a member of any one of the generaEscherichia or Pseudomonas.
 8. The recombinant cell according to claim 7wherein the recombinant cell is any one of E. coli or P. fluorescens. 9.A process for production of anabolic phloroglucinol, comprising thesteps of (A) providing (1) (a) an isolated or recombinant PhlD⁺ enzymesystem that is at least one of PhlA⁻, PhlB⁻, or PhlC⁻, or (b) a PhlD⁺recombinant cell, that is at least one of PhlA⁻, PhlB⁻, or PhlC⁻, or (c)a PhlD⁺ recombinant cell that is a PhlD⁺ cell that has been geneticallyengineered to further increase the expression of PhlD therein, theenzyme system or recombinant cell being capable of convertingmalonyl-CoA to phloroglucinol; and (2) at least one of malonyl-CoA oranother carbon source that the enzyme system or recombinant cell iscapable of converting to malonyl-CoA; and (B) contacting (1) themalonyl-CoA with the enzyme system or recombinant cell under conditionsin which it can synthesize phloroglucinol therefrom, or (2) the othercarbon source with the enzyme system or recombinant cell underconditions in which it can convert the carbon source to malonyl-CoA andcan synthesize phloroglucinol therefrom; thereby obtaining anabolicphloroglucinol.
 10. The process according to claim 9, wherein the enzymesystem (1)(a) or cell (1)(b) is PhlA⁻, PhlB⁻, and PhlC⁻.
 11. The processaccording to claim 9, wherein said enzyme system or recombinant cellcomprises a malonyl-CoA synthesis enzyme.
 12. The process according toclaim 11, where the malonyl-CoA synthesis enzyme is any one ofmalonyl-CoA synthetase, malonyl-CoA decarboxylase, or acetyl-CoAcarboxylase.
 13. The process according to claim 9, comprising providingsaid carbon source, wherein said carbon source is a simple carbonsource.
 14. The process according to claim 13, wherein said carbonsource comprises a saccharide, an aliphatic polyol, or a combinationthereof.
 15. The process according to claim 14, wherein said carbonsource comprises glucose, xylose, arabinose, glycerol, or a combinationthereof.
 16. The process according to claim 13, wherein a recombinantcell is provided and said step of contacting (B) comprises culturing thecell in a growth medium containing the carbon source.
 17. The processaccording to claim 16, wherein said culturing is performed as anextractive fermentation.
 18. The process according to claim 16, whereinsaid culturing is performed using a dual temperature profile. 19.Isolated anabolic phloroglucinol.
 20. An isolated or recombinantphloroglucinol synthase enzyme capable of converting malonyl-CoA tophloroglucinol, wherein said enzyme comprises an amino acid sequencethat is at least 90% homologous to SEQ ID NO:2.
 21. The enzyme accordingto claim 20, wherein said enzyme comprises the amino acid sequence ofSEQ ID NO:2.
 22. The enzyme according to claim 20, wherein said enzymecomprises a conservatively substituted variant of the amino acidsequence of SEQ ID NO:2.
 23. Isolated or recombinant nucleic acid vectorcomprising at least one open reading frame encoding a PhlD enzyme, andthat is at least one of phlA⁻, phlB⁻, or phlC⁻, and which furthercomprises at least one open reading frame encoding a malonyl-CoAsynthesis enzyme.
 24. The nucleic acid according to claim 23, whereinsaid nucleic acid comprises a base sequence of SEQ ID NO:1, an RNA basesequence corresponding thereto, or a codon sequence redundant therewith.25. The nucleic acid according to claim 23, wherein said nucleic acidcomprises a base sequence at least 80% homologous to SEQ ID NO:1.
 26. AphlD⁺ recombinant cell, wherein said cell has been transformed with anucleic acid vector comprising at least one open reading frame encodinga PhlD enzyme, from which the cell can express phloroglucinol synthase,the recombinant cell being phlA⁻, phlB⁻, and phlC⁻ and being capable ofconverting malonyl-CoA to phloroglucinol by action of phloroglucinolsynthase expressed therein.
 27. A process for the preparation ofresorcinol, comprising (A)(1) anabolically biosynthesizingphloroglucinol by action of (a) an isolated or recombinant PhlD⁺ enzymesystem that is at least one of PhlA⁻, PhlB⁻, or PhlC⁻, or (b) a PhlD⁺recombinant cell that is at least one of PhlA⁻, PhlB⁻, or PhlC⁻, or (c)a PhlD⁺ recombinant cell that is a PhlD⁺ cell that has been geneticallyengineered to further increase the expression of PhlD therein, theenzyme system or recombinant cell being capable of convertingmalonyl-CoA to phloroglucinol; and (2) providing hydrogen and a rhodiumcatalyst; and (B) contacting phloroglucinol biosynthesized thereby withthe hydrogen and the rhodium catalyst under conditions in which thephloroglucinol is hydrogenated to form resorcinol, thereby preparingresorcinol.
 28. A method for manufacturing a medicament, cosmetic, dye,polymer resin, rubber, adhesive, sealant, coating, composite material,or laminated or bonded material, comprising (1) anabolicallybiosynthesizing phloroglucinol by action of (a) an isolated orrecombinant PhlD⁺ enzyme system that is at least one of PhlA⁻, PhlB⁻, orPhlC⁻, or (b) a PhlD⁺ recombinant cell that is at least one of PhlA⁻,PhlB⁻, or PhlC⁻, or (c) a PhlD⁺ recombinant cell that is a PhlD⁺ cellthat has been genetically engineered to further increase the expressionof PhlD therein, the enzyme system or recombinant cell being capable ofconverting malonyl-CoA to phloroglucinol; and (2) preparing amedicament, cosmetic, dye, polymer resin, rubber, adhesive, sealant,coating, composite material, or laminated or bonded material fromanabolic phloroglucinol biosynthesized thereby, or from resorcinolobtained by chemically modifying the anabolic phloroglucinol.
 29. Amedicament, cosmetic, dye, polymer resin, rubber, adhesive, sealant,coating, composite material, or laminated or bonded material prepared bya process comprising (1) anabolically biosynthesizing phloroglucinol byaction of (a) an isolated or recombinant PhlD⁺ enzyme system that is atleast one of PhlA⁻, PhlB⁻, or PhlC⁻; or (b) a PhlD⁺ recombinant cellthat is at least one of PhlA⁻, PhlB⁻, or PhlC⁻; or (c) a PhlD⁺recombinant cell that is a PhlD⁺ cell that has been geneticallyengineered to further increase the expression of PhlD therein; theenzyme system or recombinant cell being capable of convertingmalonyl-CoA to phloroglucinol; and (2) preparing a medicament, cosmetic,dye, polymer resin, rubber, adhesive, sealant, coating, compositematerial, or laminated or bonded material from anabolic phloroglucinolbiosynthesized thereby, or from resorcinol obtained by chemicallymodifying the anabolic phloroglucinol.
 30. A process for the preparationof a recombinant cell that is capable of biosynthesizing phloroglucinolfrom malonyl-CoA, comprising (A) transforming a cell with aPhlD-encoding nucleic acid by (1) providing isolated or recombinantnucleic acid that is phlA⁻, phlB⁻, and phlC⁻ and that contains at leastone open reading frame encoding a PhlD enzyme, or contains both at leastone open reading frame encoding a PhlD enzyme and at least one openreading frame encoding a malonyl-CoA synthesis enzyme, (2) providing (a)a PhlD⁻, phlA⁻, phlB⁻, and phlC⁻ cell that can be transformed with saidnucleic acid, or (b) a PhlD⁺, phlA⁻, phlB⁻, and phlC⁻ cell that can betransformed with said nucleic acid; and (3) contacting the cell with thenucleic acid under conditions in which the cell can be transformed withthe nucleic acid, the nucleic acid being capable of expression by thecell, thereby obtaining a recombinant cell; or (B) inactivating genes ina phlD⁺ cell by (1) providing a phlD⁺ cell that is at least one ofphlA⁺, phlB⁺, or phlC⁺; and (2) inactivating all of said phlA, phlB, andphlC genes therein, thereby obtaining a recombinant cell that is phlA⁻,phlB⁻, and phlC⁻.
 31. A method for producing a propellant or explosivecompound comprising (A) anabolically biosynthesizing phloroglucinol byaction of (1) an isolated or recombinant PhlD⁺ enzyme system that is atleast one of PhlA⁻, PhlB⁻, or PhlC⁻, or (2) a PhlD⁺ recombinant cellthat is at least one of PhlA⁻, PhlB⁻, or PhlC⁻, or (3) a PhlD⁺recombinant cell that is a PhlD⁺ cell that has been geneticallyengineered to further increase the expression of PhlD therein, theenzyme system or recombinant cell being capable of convertingmalonyl-CoA to phloroglucinol; and (B) chemically modifying anabolicphloroglucinol biosynthesized thereby, or chemically modifyingresorcinol prepared therefrom, to produce a propellant or explosivecompound.
 32. The method according to claim 31 wherein said anabolicphloroglucinol is biosynthesized by a process comprising the steps of(A) providing (1) (a) said isolated or recombinant PhlD⁺ enzyme system(A)(1), or (b) said PhlD⁺ recombinant cell (A)(2), or (c) said PhlD⁺recombinant cell (A)(3), the enzyme system or recombinant cell beingcapable of converting a carbon source to phloroglucinol; and (2) atleast one of malonyl-CoA or another carbon source that the enzyme systemor recombinant cell is capable of converting to malonyl-CoA; and (B)contacting (1) the malonyl-CoA with the enzyme system or recombinantcell under conditions in which it can synthesize phloroglucinoltherefrom, or (2) the other carbon source with the enzyme system orrecombinant cell under conditions in which it can convert the carbonsource to malonyl-CoA and can synthesize phloroglucinol therefrom;thereby obtaining anabolic phloroglucinol.
 33. The method according toclaim 32, wherein said enzyme system or recombinant cell comprises amalonyl-CoA synthesis enzyme.
 34. The method according to claim 33,where the malonyl-CoA synthesis enzyme is any one of malonyl-CoAsynthetase, malonyl-CoA decarboxylase, or acetyl-CoA carboxylase. 35.The method according to claim 31, wherein the PhlD⁺ enzyme system (A)(1)or recombinant cell (A)(2) is PhlA⁻, PhlB⁻, and PhlC⁻.
 36. The methodaccording to claim 31, wherein a recombinant cell is provided.
 37. Themethod according to claim 36, wherein the recombinant cell is a phlD⁺recombinant cell that is at least one of phlA⁻, phlB⁻, or phlC⁻.
 38. Themethod according to claim 37, wherein the phlD⁺ recombinant cell isphlA⁻, phlB⁻, and phlC⁻.
 39. The method according to claim 37, whereinthe recombinant cell is produced by a process comprising providing aphlABCD⁺ cell, and inactivating at least one phlA, phlB, or phlC genethereof.
 40. The method according to claim 38, wherein the recombinantcell is produced by a process comprising providing a phlABCD⁺ cell, andinactivating all of the phlA, phlB, and phlC genes thereof.
 41. Themethod according to claim 38, wherein the recombinant cell is producedby a process comprising providing a phlABCD⁻ cell and inserting a phlDgene therein.
 42. The method according to claim 41, wherein said phlDgene is located in genomic DNA of the cell.
 43. The method according toclaim 41, wherein said phlD gene is located in extra-genomic DNA of thecell.
 44. The method according to claim 36, wherein the recombinant cellis produced by a process further comprising inserting therein a geneencoding a malonyl-CoA synthesis enzyme that is any one of malonyl-CoAsynthetase, malonyl-CoA decarboxylase, or acetyl-CoA carboxylase. 45.The method according to claim 36, wherein the recombinant cell is amicrobe.
 46. The method according to claim 45, wherein the recombinantcell is a yeast, fungus, or bacterium.
 47. The method according to claim46, wherein the recombinant cell is a bacterium.
 48. The methodaccording to claim 47, wherein the recombinant cell is a member of anyone of the genera Escherichia or Pseudomonas.
 49. The method accordingto claim 48, wherein the recombinant cell is any one of E. coli or P.fluorescens.
 50. The method according to claim 32, comprising providingsaid carbon source, wherein said carbon source is a simple carbonsource.
 51. The method according to claim 50, wherein said carbon sourcecomprises a saccharide, an aliphatic polyol, or a combination thereof.52. The method according to claim 51, wherein said carbon sourcecomprises glucose, xylose, arabinose, glycerol, or a combinationthereof.
 53. The method according to claim 32, wherein a recombinantcell is provided and said step of contacting (B) comprises culturing thecell in a growth medium containing the carbon source.
 54. The methodaccording to claim 53, wherein said culturing is performed as anextractive fermentation.
 55. The method according to claim 53, whereinsaid culturing is performed using a dual temperature profile.
 56. Anexplosive or propellant composition prepared by a process comprising (1)anabolically biosynthesizing phloroglucinol by action of (a) an isolatedor recombinant PhlD⁺ enzyme system that is at least one of PhlA⁻, PhlB⁻,or PhlC⁻; or (b) a PhlD⁺ recombinant cell that is at least one of PhlA⁻,PhlB⁻, or PhlC⁻; or (c) a PhlD⁺ recombinant cell that is a PhlD⁺ cellthat has been genetically engineered to further increase the expressionof PhlD therein; the enzyme system or recombinant cell being capable ofconverting malonyl-CoA to phloroglucinol; and (2) preparing an explosiveor propellant composition from anabolic phloroglucinol biosynthesizedthereby, or from resorcinol obtained by chemically modifying theanabolic phloroglucinol.
 57. A composition comprising isolated anabolicphloroglucinol or a derivative thereof.
 58. The composition according toclaim 57, wherein said composition is a medicament, cosmetic, dye,polymer resin, rubber, adhesive, sealant, coating, composite material,or laminated or bonded material.
 59. The composition according to claim57, wherein said composition is an explosive or propellant.
 60. Thecomposition according to claim 57, wherein said derivative is resorcinolor a derivative thereof.